Inhibition of myeloid-derived suppressor cell arginase-1 production enhances T-cell-based immunotherapy against Cryptococcus neoformans infection

Cryptococcosis is a potentially lethal disease that is primarily caused by the fungus Cryptococcus neoformans, treatment options for cryptococcosis are limited. Here, we show glucuronoxylomannan, the major polysaccharide component of C. neoformans, induces the recruitment of neutrophilic myeloid-derived suppressor cells in mice and patients with cryptococcosis. Depletion of neutrophilic myeloid-derived suppressor cells enhances host defense against C. neoformans infection. We identify C-type lectin receptor-2d recognizes glucuronoxylomannan to potentiate the immunosuppressive activity of neutrophilic myeloid-derived suppressor cells by initiating p38-mediated production of the enzyme arginase-1, which inhibits T-cell mediated antifungal responses. Notably, pharmacological inhibition of arginase-1 expression by a specific inhibitor of p38, SB202190, or an orally available receptor tyrosine kinase inhibitor, vandetanib, significantly enhances T-cell mediated antifungal responses against cryptococcosis. These data reveal a crucial suppressive role of neutrophilic myeloid-derived suppressor cells during cryptococcosis and highlight a promising immunotherapeutic application by inhibiting arginase-1 production to combat infectious diseases.

The authors have conducted an extremely comprehensive study supporting their hypothesis that Ctype lectin receptor-2d (Clec2d) recognizes GXM to potentiate the immunosuppressive activity of neutrophilic MDSCs through initiating p38-mediated production of Arg-1, which inhibits T cellmediated antifungal responses against C. neoformans infection.
I have no major criticisms of the work General comment: A grammatical review of the manuscript is needed. The Figures each show large amounts of data and the pathological sections so small that it is not possible to interpret them with respect to infiltrating cell types or constituent cells in "granulomas" or indeed if they are true granulomas.

Introduction:
The section on the potential clinical relevance of the study as summarised in the introduction needs to be improved. Refs 2 and3 (1991 and2003) need updating and qualification with respect to ART (antiretroviral) therapy Line 71. A reference for statement that efficacy of antifungal drugs is diminishing due to increased drug resistance and inability to assist the antifungal response is needed. Additional rationale for new therapeutic approaches (not mentioned) is the toxicity and need for IV therapy for the most effective drug (AMB).
In Fig 1 lung recruitment of M-MDSC and PMN-MDSC was assessed whereas blood MDSC were assessed in 15 patients with pulmonary cryptococcosis and 14 with cryptococcal meningitis. It is not stated whether any were immunosuppressed (eg by HIV or other cause) which might influence the results. Although when expressed as a ratio per 10 log 5 PBMC or as a percentage there was a statistically significant increase in PMN-MDSC especially in patients with meningitis, there was wide variation between individuals. These points should receive comment in the discussion.
In general, the different experimental manipulations in the mice significantly impacted fungal burden and pathology, to a greater extent than survival, which was prolonged rather than complete. Taken together however, the authors have shown that that in mice, PMN-MDSCs play a role in respect of amplification of cryptococcal lung disease and that the putative mechanism of action proceeds through Arg-1 as a result of Clec2d recognising components of cryptococcal GXM.
Line 324. Vandetanib is a non-specific tyrosine kinase inhibitor and it is quite a stretch to suggest that it per se is a promising immunotherapeutic for human CM. On the basis of the P38 data in particular, it is reasonable to say that inhibition of this Arg-1 pathway provides a potentially novel immunotherapeutic approach.
Overall this is a well-conducted study which adds a role for PMN-MDSCs to other mechanisms in the pathogenesis of cryptococcal disease, as has been reported in other infectious diseases such as tuberculosis.
Reviewer #2 (Remarks to the Author): Li and colleagues investigated the effect of Cryptococcus neoformans infection and glucorunoxylomannan (GXM; main capsular component) release on neutrophilic myeloid-derived suppressor cells (MDSCs). Release of GXM by the fungus increases recruitment of neutrophilic MDSCs and this is disease enhancing. Clec2d, a C-type lectin receptor, binds to GXM and promotes neutrophilic MDSCs via p38-mediated production of arginase-1 (Arg-1), which impairs T cell responses against the fungus. They also validated the pharmacological use of a p38 inhibitor, SB202190, which reduces the expression of Arg-1 and vandetanib, an inhibitor of tyrosine kinase as stimulants of T cell antifungal responses. The strengths of the study include the huge amount of work performed by the team including the generation of an Clec2d knock out mouse and the use of many techniques (e.g., flow cytometry, RNA seq, histopathology, etc.). However, the premise that C. neoformans causes disease in immunocompetent individuals is weak given that cryptococcosis affects mostly T cell deficient individuals such as those with HIV+/AIDS, organ transplantation, etc. It is uncertain how the proposed therapy will work in immunocompromised individuals with deficient cellmediated immunity. Furthermore, there are many other weaknesses in the study that are described below. 1. The number of deaths per year globally due to cryptococcal meningitis are ~200,000 not ~625,000. This is an old piece of information.
2. In addition to resistance and their inability to induce a strong immune response, antifungal drugs to treat cryptococcosis are toxic, have difficulty penetrating the CNS, and are not accessible in the world regions where are mostly needed. All this is also exacerbated by the fact that individuals with cerebral cryptococcosis are immunosuppressed. 3. Methods are poorly written to reproduce the studies or data interpretation. For example, routes of infection, mouse strains, and many other specifics to understand the studies are missing or insufficiently described. 4. Also, there is a concern with the patient data presented in Fig. 1D. The data points of the graphs look the same and have similar p values even when different parameters are analyzed (e.g., MDSCs/10^5 PBMSs vs. % MDSCs in PBMSs). If the same data is analyzed using different parameters, the graphs should be at least slightly different but not the same as presented. This reviewer gives the benefit of the doubt to the authors because unintended mistakes happen particularly when you deal with the amount of data presented in this study. Nevertheless, the methods are so poorly described that it is impossible to understand the analyses performed. Fig. 2D, the third graph to the right is missing the error bars. In fact, the statistics described in the methods are incomplete or not properly explained to understand the details of the studies. 5. Many results are logically presented. However, the discussion does not make justice to the work performed. It is inadequately described or put on perspective with previous or possible future research. 6. The work might be new in cryptococcosis, but similar work was described in Candida albicans (PMID: 25771792), therefore, these studies lack novelty because the overall connection between MDSCs and T cells is expected. In addition, it is uncertain the potential application of the therapeutics proposed in the setting of immunosuppression or in cerebral cryptococcosis, the main reason of death in patients. Also, the use of T cell depleted, or deficient mice would have been more appropriate for these studies.
We would like to thank the reviewers for their critical and constructive comments and suggestions. Based on their comments, we have performed all of the requested experiments and revised our manuscript. The following is the point-to-point responses to their comments.

Reviewer #1
The authors have conducted an extremely comprehensive study supporting their hypothesis that C-type lectin receptor-2d (Clec2d) recognizes GXM to potentiate the immunosuppressive activity of neutrophilic MDSCs through initiating p38-mediated production of Arg-1, which inhibits T cell-mediated antifungal responses against C. neoformans infection.
I have no major criticisms of the work.
General comment:

1.
A grammatical review of the manuscript is needed.
Response: Thank you for your good suggestion. A grammatical review of our revised manuscript has been made by an academic English editor of AJE company.

2.
The Figures each show large amounts of data and the pathological sections so small that it is not possible to interpret them with respect to infiltrating cell types or constituent cells in "granulomas" or indeed if they are true granulomas.

Response: We have rearranged Figure 1 and moved the part of original Fig 1A to
Supplementary S1B in our revised manuscript. Thus, the pathological sections have been zoomed in (Fig 1H and 1K). Dotted lines indicate the formation sites of

granulomas. Black arrows indicate yeast cells. We can observe that PMN-MDSC depletion by Ly-6G antibody led to a low number of yeast cells, which were mostly encapsulated in the granulomatous tissues in the infected lungs.
3. The section on the potential clinical relevance of the study as summarized in the introduction needs to be improved. 129.), they summarize that GXM contributes to immune suppression through inducing IL-10 and TGF-β production whereas GalXM can induce the production of pro-inflammatory cytokines TNF-α and IL-6. The common feature is that both

GXM and GalXM can induce apoptosis of T cells and macrophages. They
conclude that GalXM-mediated cell death could enhance the suppressive effect of GXM during cryptococcosis. We extracted GXM and GalXM from C. neoformans, which were confirmed by a commercially available antibody 18B7 (Fig R1A). We further showed that GXM was more potent to induce the tolerogenic activity of MDSCs than that of GalXM, which was evidenced by their different abilities to impair the proliferation of CD4 + and CD8 + T cells (Fig R1B).

9.
In general, the different experimental manipulations in the mice significantly impacted fungal burden and pathology, to a greater extent than survival, which was prolonged rather than complete. Taken together however, the authors have shown that that in mice, PMN-MDSCs play a role in respect of amplification of cryptococcal lung disease and that the putative mechanism of action proceeds through Arg-1 as a result of Clec2d recognising components of cryptococcal GXM.

Response: To evaluate the GXM-induced tolerogenic activity of PMN-MDSCs on the host defense against C. neoformans infection, we adoptively transferred PMN-
MDSCs sorted from the lungs of mice with or without GXM treatment and monitored their impact on fungal burdens in Cap59-infected mice (Fig R3A). We found that this transfer significantly increased fungal burdens in lungs of Cap59infected mice (Fig R3B). Moreover, we showed that only adoptive transfer of 10. Line 324. Vandetanib is a non-specific tyrosine kinase inhibitor and it is quite a stretch to suggest that it per se is a promising immunotherapeutic for human CM.

Cap59-infected WT mice (Figure 4F-G). Together, we suggest that
On the basis of the P38 data in particular, it is reasonable to say that inhibition of this Arg-1 pathway provides a potentially novel immuno-therapeutic approach.
Response: Thank you for your positive comments.

Overall this is a well-conducted study which adds a role for PMN-MDSCs to
other mechanisms in the pathogenesis of cryptococcal disease, as has been reported in other infectious diseases such as tuberculosis.
Response: Thank you for your positive comments.

Reviewer #2
Li Furthermore, there are many other weaknesses in the study that are described below.
1. The number of deaths per year globally due to cryptococcal meningitis are ~200,000 not ~625,000. This is an old piece of information.
Response: Thank you for pointing this out. We have revised this statement as following: "An estimated 223,100 cases of cryptococcal meningitis occur globally annually, which lead to about 181,100 deaths." 2. In addition to resistance and their inability to induce a strong immune response, antifungal drugs to treat cryptococcosis are toxic, have difficulty penetrating the CNS, and are not accessible in the world regions where are mostly needed.
All this is also exacerbated by the fact that individuals with cerebral cryptococcosis are immunosuppressed. 3. Methods are poorly written to reproduce the studies or data interpretation. For example, routes of infection, mouse strains, and many other specifics to understand the studies are missing or insufficiently described.

Response
Response: Thank you for your critical comments. We have added a more derail description of methods in our revised manuscript, which was highlighted with red color in the section of Methods.
4. Also, there is a concern with the patient data presented in Fig. 1D. The data points of the graphs look the same and have similar p values even when different parameters are analyzed (e.g., MDSCs/10^5 PBMSs vs. % MDSCs in PBMSs). If the same data is analyzed using different parameters, the graphs should be at least slightly different but not the same as presented. This reviewer gives the benefit of the doubt to the authors because unintended mistakes happen particularly when you deal with the amount of data presented in this study.
Response: Thank you for pointing it out. Actually, we made a mistake about p value of analyzing MDSCs number in patient data. We have corrected them and moved original part of Fig. 1D to Supplementary Fig 1E. 5. Nevertheless, the methods are so poorly described that it is impossible to understand the analyses performed. Fig. 2D, the third graph to the right is missing the error bars. In fact, the statistics described in the methods are incomplete or not properly explained to understand the details of the studies. Fig 2B, 2D and 5M. We also added the description of statistics in the section of Methods as following: "data was expressed as mean ± SEM, Differences between two groups were compared using Mann-Whitney (GraphPad Prism, La Jolla, CA, USA), and grouped comparison was evaluated by non-parametric ANOVA and subsequent Kruskal-Wallis (Kruskal-Wallis). Statistical significance was set on the basis of P value.

Response: We have added the missing error bars in
n.s., *P < 0.05, **P < 0.01, ***P < 0.001." 6. Many results are logically presented. However, the discussion does not make justice to the work performed. It is inadequately described or put on perspective with previous or possible future research.
Response: Thank you for your good suggestion, we have modified our discussion to add more detail description and perspectives in our revised manuscript, which were highlighted with red color.

The work might be new in cryptococcosis, but similar work was described in
Candida albicans (PMID: 25771792), therefore, these studies lack novelty because the overall connection between MDSCs and T cells is expected.
Response: In this previous study, they show that beta- 8. In addition, it is uncertain the potential application of the therapeutics proposed in the setting of immunosuppression or in cerebral cryptococcosis, the main reason of death in patients. Also, the use of T cell depleted, or deficient mice would have been more appropriate for these studies.
Response: Thank you for your critical comments. According to your suggestions, we used anti-CD4 antibody to deplete CD4 + T cells in murine model of C.
neoformans infection (Fig. R4A). Although we observed that the depletion of

CD4 + T cells significantly increased the percentage of CD8+T cells in infected lungs,
this depletion significantly increased fungal burden in lungs of infected mice (Fig.   R4B). Moreover, CD4 + T cell depletion completely impaired the effects of vandetanib treatment on reducing fungal burden of infected lungs (Fig. R4B).
Together, these data confirmed that therapeutic efficacy with vandetanib against

C. neoformans infection was dependent on CD4 + T cells. We have also added your
suggested comments to the Discussion section in our revised manuscript.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): The authors have satisfactorally answered most of my queries and have amended the manuscript accordingly.
The following still need to be addressed.
Original issue 4. Refs 2 and 3 (1991 and 2003) need updating and qualification with respect to ART (antiretroviral) therapy.

Response
We have updated references according to your suggestion and added new statement with respect to ART as following: The effectiveness of antiretroviral therapy (ART) in restoring CD4+ T cell numbers in AIDS patients has dramatic reductions in mortality and morbidity of cryptococcal meningitis4 However, severe adverse reaction to ART is referred to as immune reconstitution inflammatory syndrome, which is characterized by tissue-destructive inflammation and arises as CD4+ T cells re-emerge5. Therefore, there is an urgent need to develop new immunotherapeutic strategies for restoring the function of CD4+ T cells to combat cryptococcosis.

Comment
The logic of the additional sentences is confusing to me. Active cryptococcal meningitis has a significant risk of causing IRIS in patients with AIDS and low CD4 counts if ART therapy is commenced too early (before the fungal infection has been controlled). Although the authors assert that "there is an urgent need to develop new immunotherapeutic strategies for restoring the function of CD4+ T cells to combat cryptococcosis" the manuscript is about "enhanced T cell-mediated antifungal responses against C. neoformans infection by an effect on existing CD4 cells. The problem in HIV-infected patients who make up the majority of those with CM globally is that their CD4 cells are low to very low and may not therefore be able to respond to the immunotherapeutics under consideration. This paragraph needs rewording.
Having said this, notably, especially, in resource rich countries, other causes of immunosuppression and additional risk factors, not necessarily associated with low CD4 counts/function are importantand responses to the immune-therapeutics may be advantageous for CM. There is a hint that responses of the immunosuppressed and non-immunosuppressed human subjects are similar in the study (although the sample sizes are very small).

P5 line 130
Start results of the human studies on a new line. Suggest re-word to clarify that all 29 patients were HIV-negative and give the total numbers of immunosuppressed and non-immunosuppressed with PC and CM in the text. The differences between immunosuppressed or would be fine in fig (as this information is highly relevant to clinical context) but low sample size needs a comment.
Discussion: lines 432-437 need to qualify the statement by specifying that there were small numbers of immunosuppressed and non-immunosuppressed human subjects. I note that the lack of information in the context of HIV-associated CD4 deficiency has been commented on (and indeed it should be stated that this may prevent the immunotherapy strategy working).
The real strength of the study is the biology rather than likely therapeutic applications. Grammatical edits will be needed if accepted.
Reviewer #2 (Remarks to the Author): My comments were diligently addressed by the authors.
Reviewer #3 (Remarks to the Author): I did not review this paper before, so I carefully examined both manuscript and authors response. I believe, although overall concept of the study is not novel, the specific information regarding Glec2d regulation of MDSC function is novel and interesting. Experiments are well performed. Although mechanism linking p38 and Glec2d as well as p38 and arginase expression remained unclear, this can be developed in subsequent studies.
The main problem with the study is that authors did not provide direct evidence that cells they observed in their model (Figure 1) are indeed MDSC. Infection would cause mobilization of classical neutrophils so this is rather important question. Surface markers used by the authors cannot discriminate between PMN and PMN-MDSC as was demonstrated in the papers authors referred to. So functional evidence of suppression would be important. At minimum, detailed gene expression profile confirming that pathological nature of these cells. Authors used suppression assay later in the paper where they assess the effect of GXM. However, it was done with in vitro generated rather artificial cells (GM-CSF+IL-6), which underscore need for some ex vivo experiment.
The other issue that need correction is the fact that in Figure 4 authors observed only partial abrogation of suppressive activity in KO cells. This fact was not explained. It seems that presented mechanism only partially responsible for the observed phenomenon.